A Novel Lectin with Antiproliferative and HIV-1 Reverse Transcriptase Inhibitory Activities from Dried Fruiting Bodies of the Monkey Head Mushroom Hericium erinaceum

A lectin designated as Hericium erinaceum agglutinin (HEA) was isolated from dried fruiting bodies of the mushroom Hericium erinaceum with a chromatographic procedure which entailed DEAE-cellulose, CM-cellulose, Q-Sepharose, and FPLC Superdex 75. Its molecular mass was estimated to be 51 kDa and its N-terminal amino acid sequences was distinctly different from those of other isolated mushroom lectins. The hemagglutinating activity of HEA was inhibited at the minimum concentration of 12.5 mM by inulin. The lectin was stable at pH 1.9–12.1 and at temperatures up to 70°C, but was inhibited by Hg2+, Cu2+, and Fe3+ ions. The lectin exhibited potent mitogenic activity toward mouse splenocytes, and demonstrated antiproliferative activity toward hepatoma (HepG2) and breast cancer (MCF7) cells with an IC50 of 56.1 μM and 76.5 μM, respectively. It manifested HIV-1 reverse transcriptase inhibitory activity with an IC50 of 31.7 μM. The lectin exhibited potent mitogenic activity toward murine splenocytes but was devoid of antifungal activity.

A polysaccharide with antitumor activity [34], and a laccase [35] have been reported from H. erinaceum. From the fresh fruiting bodies of H. erinaceum, a sialic acid-binding lectin (HEL) with a blocked N-terminal and a molecular mass of approximately 54 kDa has been isolated [17]. The cultures or extracts of H. erinaceum processed into tablets have been put into production on a large scale, mainly for curing gastric ulcer and chronic gastricism [36].
In the present study, we isolated and characterized a novel lectin from the dried fruiting bodies of H. erinaceum. It possessed a relatively high heat stability and pH stability, a unique amino acid N-terminal amino acid sequence, potent mitogenic activity toward murine spleen cells, reverse transcriptase HIV-1 inhibiting activity and antipoliferative activity on tumor cells. In particular, this lectin is different from HEL in some aspects. Since H. erinaceum is used in traditional Chinese medicine, the results of the present study would provide a scientific basis for the medicinal use of this mushroom.

Purification Scheme.
Dried fruiting bodies (20 g) of the H. erinaceum were homogenized in 150 mM NaCl (25 ml/g) using a Waring blender and then soaked in 500 ml of 150 mM NaCl for 12 hours. The slurry was then centrifuged at 8000 × g for 15 minutes. Afterward (NH 4 ) 2 SO 4 was added into the supernatant to 80% saturation. The precipitate was collected by centrifugation (8000 × g, 4 • C, 15 minutes), and dissolved in a small amount of distilled water and dialyzed until the final concentration of 10 mM phosphate-buffered saline (pH 7.0) was attained. The crude extract was then applied to a column of DEAE-cellulose column (Sigma, 1.0 cm × 15 cm) which had been previously equilibrated with 10 mM phosphate-buffered saline (pH 7.0). Following elution of the unadsorbed fraction D1 with the starting buffer, fractions D2 and D3 were obtained by eluting the column with 50 Mm NaCl and 300 mM NaCl in the phosphatebuffered saline respectively. The active fraction (D3) was applied to a CM-cellulose column (Sigma, 1.0 cm × 15 cm) in 10 mM NH 4 OAc buffer (pH 5.1). After removal of the unadsorbed protein with the starting buffer, the column was eluted with 50 mM NaCl in the starting buffer to attain the active fraction C2. Subsequently it was further fractionated on an ion exchange chromatography Q-Sepharose column (Sigma, 0.5 × 10 cm). After the unadsorbed fraction (Q1) had been eluted in 10 mM NH 4 OAc buffer (pH 5.1), the adsorbed fractions were eluted with a linear gradient of 0-400 mM NaCl in the same buffer. The active peak (Q3) was subjected to final purification on a Superdex G-75 HR 10/30 column by fast protein liquid chromatography using an AKTA Purifier (GE Healthcare, US) and was eluted with 10 mM phosphate buffer (pH 7.5) containing 150 mM NaCl. Peak SU1 represented the purified lectin (HEA).

Determination of Molecular Mass and N-Terminal
Sequence. The purified lectin was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for molecular mass determination following with the procedure of Laemmli and Favre (1973) [37]. Gel filtration on a performed Superdex 75 HR 10/30 column (GE Healthcare, US), which had been calibrated with molecular mass markers, was also obtained for the molecular mass determination of the lectin. The N-terminal sequence of the lectin was obtained by using a Hewlett-Packard HP G1000A Edman degradation unit and an HP 1000 HPLC System [37].

Assay of Lectin (Hemagglutinating) Activity.
In the assay for lectin (hemagglutinating) activity, a serial twofold delution of the lectin solution in microtiter U-plates (50 μl) was mixed with 50 μl of 2% suspension of rabbit red cells in phosphate-buffered saline (pH 7.2) at 20 • C. The results were recorded after about 1 hour when the blank had fully sedimented. The hemagglutintion titer, defined as the reciprocal of the highest dilution of the samples exhibiting hemagglutination, was reckoned as one hemagglutination unit. Specific activity is the number of hemagglutination units per mg protein [11].
The hemagglutination inhibition tests to investigate inhibition of lectin-induced hemagglutination by various carbohydrates were executed in a way similar to the hemagglutination test. The carbohydrates examined comprised inulin, D-melibiose, D-fructose, L-arabinose, L-rhamnose, D-xylose, L-sorbose, inositol, lactose, D-galactose, sorbose, D-arabinose, adonitol, cellobiose, D-glucose, D-mannose, raffinose, turanose, sucrose, maltose, and dulcitol. Serial twofold dilutions of sugar samples were prepared in phosphate buffered saline. All of the dilutions were mixed with an equal volume (25 μl) of a solution of the lectin with 32 hemagglutination units. The mixture was permitted to stand for 30 minutes at room temperature and then mixed with 50 μl of a 2% rabbit erythrocyte suspension. The minimum concentration of the sugar in the final reaction mixture which completely inhibited 32 hemagglutination units of the lectin preparation was computed [11].
The effects of NaOH, HCl, metal chlorides, and temperature on hemagglutininating activity of the lectin were studied as previously described in [11,12].

Assay of Antiproliferative Activity on Tumor Cell Lines.
The tumor cell lines, human breast cancer (MCF7) and hepatoma (HepG2), were acquired from American Type Culture Collection (ATCC). They were kept in Dulbecco modified Eagl's medium (DMEM) containing 10% fetal bovine serum (FBS), 100 mg/L streptomycin and 100 IU/ml penicillin at 37 • C in a humidified atmosphere of 5% CO 2 . Cells (1 × 10 4 ) in their exponential growth phase were seeded into each well of a 96-well culture plate (Nunc, Denmark). Incubation was carried out for 3 hours before addition of the lectin. Incubation was then continued for another 48 hours. Radioactive precursor, 1 μCi, ([ 3 H-methyl] thymidine, from GE Healthcare) was then added to each well and incubated for 6 hours. The cultures were then harvested by means of a cell harvester. The incorporated radioactivity was determined by liquid scintillation counting [44].

Assay for HIV-1 Reverse Transcriptase Inhibitoryactivity.
The inhibitory activity towards human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) was evaluated by using an enzyme-linked immunosorbent assay (ELISA) kit from Boehringer Mannheim (Germany). The assay makes use of the ability of reverse transcriptase to synthesize DNA, commencing from the template/primer hybrid poly(A)-oligo(dT) 15 . The digoxigenin-and biotinlabeled nucleotides in an optimized ratio are incorporated into the DNA molecule synthesized by the RT. The detection and quantification of the synthesized DNA as a measure of RT activity follows sandwich ELISA protocol. A fixed amount (4-6 ng) of recombinant HIV-1 RT was used. The inhibitory activity of the lectin was expressed as percent inhibition as compared to a control without the protein [45].

Assay of Mitogenic Activity Toward Mouse Splenocytes.
Four C57BL/6 mice (20-25 g) were sacrificed by cervical dislocation and the spleens were aseptically excised. Splenocytes were isolated by pressing the tissue through a sterilized 100mesh stainless steel sieve and respended to 5 × 10 6 cells/ml in RPMI 1640 culture medium containing 10% fetal bovine serum, 100 units penicillin/ml, and 100 μg streptomycin/ml. The splenocytes (7 × 10 5 cells/0.1 ml/well) were seeded into a 96-well culture microplates and a serial dilution of the lectin in 100 μl medium was added. Following incubation of the splenocytes at 37 • C in a humidified atmosphere of 5% carbon dioxide in the presence or absence of the lectin for 24 hours, the cells in one well were pulsed with 10 μCi [ 3 H-methyl] thymidine (specific activity 0.25 μCi/mmol, GE Healthcare) was added, and the splenocytes were incubated for another 6 hours under the same conditions, and then harvested onto a glass fiber filter using an automated cell harvester. The radioactivity was counted using a Beckman model LS 6000SC scintillation counter. The proliferative (mitogenic) response was expressed as mean counts per minute (cpm) [44]. All reported values are the means of triplicate samples.

2.7.
Assay of Antifungal Activity. The assays were conducted as detailed by Wang and Ng [46]. The assay for inhibitory activity toward the fungi Fusarium oxysporum, Rhizoctonia cerealis, Rhizoctonia solani, and Sclerotinia sclerotiorum was carried out in 100 mM × 15 mM petri dishes containing 10 ml of potato dextrose agar (PDA). After formation of the mycelial colony, sterile blank paper disks (0.625 cm in diameter) were deposited at a distance of 0.5 cm away from the circumference of the mycelial colony. An aliquot (15 μl) of the lectin was added to a disk. The dishes were incubated at 25 • C for 72 hours until mycelial growth had encircled the disks containing the control and had produced crescents of inhibition around disks containing samples with antifungal activity.

Isolation and Purification of HEA.
Hemagglitinating activity in the fruiting body extract was adsorbed successively on DEAE-cellulose, CM-cellulose, and Q-Sepharose columns. Activity was located in peaks D3, C2, and Q3 (Figures 1(a), 1(b) and 1(c)). The purified fraction SU1 was obtained through gel filtration on a Superdex G-75 HR 10/30 column (Figure 1(d)). The yields and specific hemagglutinating activities of the various chromatographic   Table 1. The purified lectin appeared as a single band with a molecular mass of 51 kDa in SDS/PAGE ( Figure 2) and a peak (SU1) with a molecular mass of 51 kDa in FPLC gel filtration. The N-terminal sequence of the HEA was AFGQLSFANLAAADF, different from the other mushroom lectins shown in Table 2. A blast search revealed that there was only slight resemblance to other previously reported lectins.
HEA isolated in the present investigation differed from lectins purified from other mushrooms. HEA was adsorbed on DEAE-cellulose and CM-cellulose, and eluted with 300 mM NaCl and 50 mM NaCl, respectively. It was deadsorbed from Q-Sepharose column with a linear NaCl concentration gradient (0 to 400 mM). Compared with HEA, another H. erinaceum lectin HEL was adsorbed DEAE-Toyopearl column and Mono-S column. HEL could be also purified from the ammonium sulfate precipitate by affinity chromatography on BSM-or asialo-BSM-Toyopearl. But, recovery of the activity by affinity chromatography was much lower than that of this study procedure (10% and 8.7%, resp.) [17]. What is more, in each step a substantial amount of protein devoid of hemagglutininating activity was eliminated, indicating that the procedure was an effective one. Different from HEL which is composed of two different subunits with a molecular mass of 15 kDa and 16 kDa, HEA is monomeric with a molecular mass approximating 51 kDa. HEA displayed an N-terminal sequence with little resemblance to some of the published mushroom lectins, such as those from Agrocybe aegerita, Laccaria   Identical amino acid residues are highlighted in boldface and underscored.

Biological Characteristics of HEA.
The specificities of carbohydrate-binding of HEA were examined by hemagglutination-inhibition assay. The hemagglutinating activity of HEA (32 units) was totally inhibited by inulin at a concentration of 12.5 mM (data not shown). Carbohydrate specificity was an important characteristic of lectins. It is interesting that only inulin, a plant polysaccharide, was able to inhibit the hemagglutinating activity of HEA. To date several inulin-specific lectins have been reported [14,47,48]. Hence HEA may be used in the production of immobilized lectin for affinity chromatography.

Other Biological Activities of HEA.
Lectins from A. bisporus, P. ostreatus, T. mongolicum, and V. volvacea exhibit antitumor activity in vivo or antiproliferative activity in vitro [5,11,23]. A. luteo-virens was devoid of antiproliferative activity on HepG2 cells [14]. HEA inhibited the proliferation of HepG2 and MCF7 tumor cells with an IC 50 value of 56.1 μM and 76.5 μM, respectively ( Figure 3). The potent antiproliferative activity of HEA is remarkable and hopefully it can be developed into an aided agent for cancer therapy.
To date very few lectins have been reported with antifungal activity and the number of lectins claimed to have antifungal activity is also relatively small [50][51][52][53]. The lectin lacked antifungal and ribonuclease activities (data not shown). HIV-RT is a key enzyme of the HIV life cycle. Screening of HIV-RT inhibitors is currently a strategy to search for anti-HIV drugs. It is possible that the mechanism of inhibition is analogous to the protein-protein interaction involved in the inhibition of HIV-1 reverse transcriptase by the homologous protease [54]. It is worth mentioning that HEA manifested a weak potent inhibitory activity toward HIV-1 RT (IC 50 = 31.7 μM, Figure 4), compared with other lectins such as S. commune lectin (IC 50 = 1.2 μM), P. citrinopileatus lectin (IC 50 = 0.93 μM) and P. adiposa lectin (IC 50 = 1.9 μM), respectively [13,44,48]. However, The P. ostreatus lectin and G. capense lectin lacked any inhibitory effect on HIV-1 reverse transcriptase [9,11]. It manifested potent mitogenic activity toward murine spleen cells, producing a 35.8-fold stimulation over the control value at at a dose of 20 μM ( Figure 5). The mitogenic activity of HEA toward murine splenocytes is in accordance with reports on lectins from the mushrooms Volvariella volvacea [8,55] and Agrocybe cylindracea [9].

Comparison with HEA and HEL.
A comparison with HEL [17] and HEA (this study) is listed in Table 6. Nterminal sequence and many biological activities, including antifungal activity, antiproliferative activity, mitogenic activity and HIV-1 RT inhibitory activity have been determined in HEA. The N-terminal sequence, antifungal activity, antiproliferative activity and HIV-1 RT inhibitory activity of HEL has not been demonstrated. However, the molecular mass of HEL is different from HEA. HEL has a molecular mass in the vicinity of 54 kDa and composed of two different subunits with a molecular mass of 15 and 16 kDa, while HEA in this study was monomeric and had a molecular mass about 51 kDa. The two lectins have very similar the thermostability. The pH stability of HEA (pH 1.9-12.1) is much higher than that of HEL (pH 5.0-10.5). The hemagglutinating activity of the HEA but not that of HEL was inhibited by Cu 2+ , Fe 3+ , and Hg 2+ ion. HEA and HEL had distinctive sugar specificities. HEL could interact with N-glycolylneuramic acid, N-acetylneuramic acid, 3 -N-acetylneuramin-lactose and galacturonic acid, while HEA was only influenced by inulin.

Conclusions
In summary, a novel lectin named HEA was isolated from dried fruiting bodies of the mushroom. It possessed a distinctive N-terminal sequence, carbohydrate specificity, and potent antiproliferative activity toward tumor cell lines, potent mitogen activity toward splenocytes and HIV-1 RT inhibitory activity. It represents an addition to the existing list of mushroom lectins.

8
Journal of Biomedicine and Biotechnology